| Background and aimsColorectal cancer(CRC) is the third cancer in the world,and it is also the third reason of tumor matality with1million new cases and more than0.5million death in one year.In recent years,with the development of life style,diet construction,environmental factor and so on, the colorectal incidence and motality increased every year in China. So it is important to study the mechanisms of colorectal cancer carcinogenesis and development, and new method to diagnose and treat colorectal cancer seems hopeful with the discovery of microRNAs.microRNAs(miRNAs) are single-stranded non-coding small RNAs of21-25nt that are highly conserved in evolution. In animals,miRNAs negatively regulate gene expression through uncompletely pairing with target messenger RNAs and play important roles in regulation of gene expression.miRNAs in animals pair with target RNAs uncomletely,and one miRNA can regulate different target mRNAs with no effect on target RNAs’integrity.So the change of one miRNA might effect many protein translation and signal pathway.More than1000miRNAs has been discovered and they could regulate about30%gene in human by targetting different mRNAs.MiRNAs regulate cells’growth,proliferation,differentiation,apoptosis and so on.Calin et al first reported that miRNAs were correlated with cancer.They found miR-15and miR-16located in tumor suppressor gene of chromosome13,reduction or deletion of both were discovered in most (about68%)of Chronic Lymphocyte Leukemia(CLL),which meaned that both were related to CLL.Then correlation between miRNAs and tumor studied further and many studies suggest that miRNAs could be a new method for tumor screening,diagnosing,prognosing and chemotherapy predicting.Discovery and study of miRNAs provide new method to elucidate the complicated gene net,it also provide new rationale for cancer screening,diagnosis and treatment.MiR-143and miR-145were firstly discovered in CRC in2003by Michael,and now more than100miRNAs has been studied in CRC which are related to carcinogenesis,development,diagnose and therapy of CRC through regulating target mRNAs. In previous study,we detected miRNAs in4paired tumor and para-tumor normal tissues by microarray and found that64miRNAs were differently expressed (more than3fold) in CRC tissues compared to para-tumor normal tissues.30miRNAs such as hsa-miR-16, hsa-miR-106a and hsa-miR-20a highly expressed.34miRNAs decreased significantly, which included mmu-miR-1224,hsa-miR-1298and hsa-miR-1275.Though hsa-miR-16and hsa-miR-106a had been studied in diffferent diseases,but they are rarely investigated in CRC.So we choose miR-16(10folds), and miR-106a (4folds) for RT-PCR verification,clinical analysis and function exploration.This study detected expresssion of miR-16and miR-106a in52CRC and para-cancer normal tissues.,the correlation between miRNAs and TNM stage,lymphonode metastasis and tissue differentiation had also be examined.Then,the cell function of miR-16and miR-106a had been explored.Last,the mechanisms of miR-16effect on apoptosis had been studied. All the research done in this study was to provide new theories and method to molecular treatment of CRC.Mehods and results 1. The correlation between miR-16,miR-106a and clinicalpathology1.1miR-16was downregulated in CRC tissues and related to tissue differentiation.Total RNA was extracted from52paired CRC and para-tumor normal tissues collected from surgery.cDNA were synthesized by TaqMan MicroRNA Reverse Transcription Kit and miR-16RT primer(Applied Biosystems,USA). Real time PCR was used to detect relative expression of miR-16in CRC tissues compared to normal tissues.Therefore, the value of the relative expression ratio<1.0was considered as low-expression in cancer relative to the non-tumorous control and vice versa.The we analyzed the association between miR-16expression and clinicopathological characteristics. We found low expression of miR-16in most of CRC tissues compared to normal tissues.Among52patients with CRC,35(67%) cases showed low-expression of miR-16(Z=-2.025,P=0.043).miR-16expression was relative to tissue differentiation (T=3.323,P=0.008),the lower expression of miR-16was associatied with lower tissue differentiation.There is no correlation between miR-16and gender,age,TNM stages and lymphode metastasis(P>0.05).1.2miR-106a are highly expressed in CRC tissues compared to para-tumor normal tissus,and both are related to TNM stages and lymphonode metastasis.Relative miR-106a expression in CRC tissues compared to normal tissues was detected by TaqMan MGB(minor groove binder)probe.We analyzed the association between the two microRNAs and clinicopathological characteristics.We found that miR-106a were expressing highly in CRC tissues. miR-106a was also increased in38CRC tissues (73%, Z=-3.748,P=0.000).The mean fold change of miR-106a in CRC tissues compared to para-tumor normal tissues were3.1.Higher expression of miR-106a were correlated to higher TNM stages(T=2.813,P=0.003respectively).miR-106a were also related to lymphode metastasis(T=-2.635,P=0.008 respectively),and the mean levels of miR-106a in lymphode positive and negative were1.29vs4.94respectively,which means that tissues of lymphode positive expressed high level of miR-106a. miR-106a expression were not correlated to gender,age and tissue differentiation(P>0.05).2The effect of miR-16and miR-106a on proliferation and apoptosis of CRC cells.2.1The effect of miR-16on cell proliferation and apoptosis2.1.1miR-16inhibited cell proliferation of HCT116and LOVO cells,but not SW480and SW620cells.HCT116,LOVO,SW480and SW620cells were choosed for their expression of wild and mutant p53gene. Four cell lines were transiently transfected inhibitors,negative control and miR-16mimics.72h after transfection CCK-8examined cell proliferation in differently groups.We found that miR-16inhibited proliferation of HCT116and LOVO cells (F=53.611,P=0.000and F=78.086,P=0.000),but not SW480and SW620cells(both P>0.05).The results suggested miR-16inhibited cell proliferation related to p53status.2.1.2miR-16induced G0/G1cell arrest through inhibiting Cycliln D1and CDK6expression.HCT116and LOVO cells were were transiently transfected inhibitors,negative control and miR-16mimics.72h after transfection PI cell cycle assay detected cell cycle and Western blot examined expression of Cyclin Dl and CDK6in differently groups.We found that miR-16arrested cell cycle at G0/G1stage(F=37.025,P=0.000and F=17.478,P=0.003respectively) and miR-16inhibited expression of Cycliln D1and CDK6.The results indicated that miR-16inducing G0/G1cell arrest through inhibiting Cycliln D1and CDK6expression,which had effect on cell proliferation.2.1.3miR-16induced apoptosis of HCT116and LOVO cells,but not SW480 and SW620cells.HCT116,LOVO,SW480and SW620cells were were transiently transfected inhibitors,negative control and miR-16mimics.72h after transfection, Annexin V and7-AAD apoptosis assay examined cell apoptosis.We found d that miR-16induced apoptosis of HCT116and LOVO cells (F=85.387,P=0.000and F=53.493,P=0.000respectively),but not SW480and SW620cells.The results suggested miR-16induced cell apoptosis related to p53status too.2.2The effect of miR-106a on proliferation and apoptosis in CRC cells.2.2.1HCT116cells were non-transfected, transiently transfected negative control and miR-106a mimics.72h after transfection,CCK-8was used to detected cell proliferation in different groups.We found that miR-106a significantly decreased cell proliferation in HCT116cells(F=114.484,P=0.000)2.2.2miR-106a induced apoptosis of HCT116cellsHCT116cells were non-transfected, transiently transfected negative control and miR-106a mimics.72h after transfection, Annexin V and7-AAD apoptosis assay examined cell apoptosis.We found d that miR-106a induced apoptosis of HCT116cells (F=30.963,P=0.000)3. Michanisms exploration of miR-16on cell apoptosis3.1miR-16increased apoptosis of HCT116and LOVO cells through survivin pathwayHCT116and LOVO cells were non-transfected, transiently transfected negative control and miR-16mimics.72h after transfection protein extracted and western blot examined expression of surviving,caspase9,caspase8and caspase3.We found that miR-16obviously decreased expression of survivin,and induced cleaved capase9and caspase3,but not caspase8.Our results suggested that miR-16induced apoptosis through survivin pathway,which could further demonstrated effect of miR-16on apoptosis.3.2survivin is one of the target of miR-16HCT116and LOVO cells were non-transfected, transiently transfeeted negative control and miR-16mimics.48h after transfection,total RNA extracted and cDNA was synthesized,real time RT-PCR was used to detected mRNA level of survivin.We found that miR-16significantly inhibited expression of survivin mRNA in HCT116and LOVO cells (F=15.607,P=0.017and F=14.891,P=0.018respectively).MiR-16inhibited expression of survivin protein as show in results above.Luciferase reporter assay was used to detected miR-16directly binding to survivin and inhibiting expression of survivin in HCT116cells.There are four groups: group negative+wild survivin3’UTR,group negative control+mutant survivin3’UTR,group miR-16+wild survivin3’UTR and group miR-16+mutant survivin3’UTR.HCT116cells cotransfected siRNA and survivin plasmid as group above and48h after transefecion,relative luciferase expression was detected in four groups.We found that luciferase activity were differently expressed in groups (F=67.952,P=0.000),and compared to negative control group, miR-16mimics significantly inhibited luciferase activity of wild Survivin,but not mutant Survivin.The results indicated that miR-16directly inhibited expression of survivin in both mRNA and protein level,survivin is one of the target of miR-16.3.3p53regulated expression of survivin by miR-16pathway.HCT116and LOVO cells were transiently transfeeted negative control and p53siRNA.48h after transfection, RNA extracted and cDNA was synthesized,real time RT-PCR was used to detected expression of miR-16.72h after transfection,protein was extracted and western blot was used to examined p53, survivin cyclinD1and CDK6expression. We found that miR-16expression in cells of p53siRNA were downregulated compared to negative control (r=17.421,P=0.000' t=7.142,P=0.019).With downregulation of p53and miR-16,survivin was upregulated,so as cyclinDl and CDK6. The results above demonstrated that miR-16,a molecular of down stream of p53,participated p53/survivin pathway.ConclusionWe could draw conclusions from the results above as follows:1、miR-16expressed low in CRC tissues compared to non-tumor tissues and miR-16expression was correlated to tissue differentiation;2、miR-106a highly expressed in CRC tissues compared to non-tumor tissues and both were related to TNM stages and lymphonode metastasis;3、miR-16inhibited cell proliferation,induced cell apoptosis,and the effect on proliferation and apoptosis was related to p53status;4、miR-106a inhibited cell proliferation and induced cell apoptosis;5、miR-16induced cell apoptosis in HCT116and LOVO cells through survivin pathway;6、miR-16decreased expression of survivin both mRNA and protein level,and survivin was one of the target of miR-16;7、p53regulated survivin expression through miR-16pathway. |
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